摘要
目的:建立UPLC/Q-TOF-MS法鉴定市售凝血酶冻干粉中的蛋白组分并进行定量。方法:样品经胰蛋白酶消化后的多肽混合物,用超高效液相色谱进行分离,并在线进入ESI—Q—TOF—MS检测,获得的肽段一级母离子和碎片离子的高分辨准确质量数据(MS^E方法),经ProteinLynx Global Server(PLGS2.4)软件蛋白质谱库检索,确定其中的蛋白组分,再以Hi3非标记蛋白定量方法对各蛋白组分进行定量,得到质量百分比定量结果。结果:在以猪血为原料生产的26批凝血酶冻干粉中,鉴定出人血白蛋白和21种猪源蛋白,其中,有1家企业的3批样品中鉴定出猪和人血浆成分,其余23批次样品均为猪血浆成分;样品中的猪凝血酶含量在1.9%~43.7%之间。在以牛血为原料生产的3批凝血酶冻干粉中共鉴定出17种牛源蛋白;样品中的牛凝血酶含量在49.4%~77.6%之间。结论:UPLC/Q-TOF—MS法灵敏度高,选择性好,通过一次分析可对凝血酶冻干粉中蛋白组分同时进行定性和定量,并可获得有关工艺相关的数据信息如来源和种属等,可用于本品的工艺研究以及质量控制研究。该法可以作为同类生化药物质量研究和组分分析的方法。
Objective:To establish an assay with high sensitivity and specificity for identification and quan- tification of protein components in commercial thrombin lyophilized powder with UPLC/Q-TOF-MS system, nethods:Lyophilized thrombin samples were digested with trypsin and then separated and analyzed with ultra-performance liquid instrument coupled with a quadrupole time-of-flight mass spectrometer. Both peptide parent ions and fragments were collected simultaneously by alternation between low and elevated energy of the collision cell( MSE methodology). The MSE data was processed by ProteinLynx Global Server (PLGS 2.4),to identify and quantify protein components in the samples. Quantization was achieved with a proven label-free quantification technique ("Hi3" method). Results:Twenty-one porcine protein groups and human albumin were identified from the pig- blood-source thrombin lyophilized powders, with thrombin content ranging from 1. 9% to 43. 7% in different manufacturers' products. Seventeen bovine protein groups were identified from the bovine-blood-source thrombin ly- ophilized powders,with thrombin content ranging from 49.4% to 77.6%. Conclusion:This assay provides valuable information to understand the origins of the samples, protein components and percentages of lyophilized thrombin powders,as well as some process-related information. The assay potentially can be used as a generic method for the quality research of similar biologics.
出处
《中国新药杂志》
CAS
CSCD
北大核心
2014年第11期1321-1325,1329,共6页
Chinese Journal of New Drugs